Activity of Alkaloids on Peptic Ulcer: What’s New?

Peptic ulcer is a common disease characterized by lesions that affect the mucosa of the esophagus, stomach and/or duodenum, and may extend into the muscular layer of the mucosa. Natural products have played an important role in the process of development and discovery of new drugs, due to their wide structural diversity and present, mostly specific and selective biological activities. Among natural products the alkaloids, biologically active secondary metabolites, that can be found in plants, animals or microorganisms stand out. The alkaloids are compounds consisting of a basic nitrogen atom that may or may not be part of a heterocyclic ring. This review will describe 15 alkaloids with antiulcer activity in animal models and in vitro studies.


Introduction
Peptic ulcer disease (PUD) is an illness characterized by lesions in the gastrointestinal mucosa that may penetrate the muscularis layer. Stomach lesions are located preferentially along the small curvature in the transition zone between the body and the antrum, whereas in the duodenum those lesions are located in the bulb or duodenal bulb [1]. PUD affects approximately 10% of the population OPEN ACCESS worldwide [2] and represents not only costs to the individual, but also for public health [3]. Both gastric and duodenal ulcers, affect men more than women, being the most prevalent the duodenal one in younger people, while gastric ulcer is more prevalent in the elderly [4].
The development of peptic ulcer is complex and involves a multifactorial process, which occurs by an imbalance between aggressive and defensive factors of the gastric mucosa [5]. The gastric mucosa is continuously exposed to harmful substances and factors, whether endogenous as acid secretion, peptic activity and biliary secretion or exogenous as the presence of Helicobacter pylori, the use excessive of alcohol, tobacco and anti-inflammatory non-steroidal (NSAIDs), besides stressful life habits [5,6].
However, despite this assault, under normal conditions the gastric mucosa is able to resist damage from 0.1 mol/L HCl and pepsin present in the gastric lumen by a variety of defense mechanisms which include as a first line the defense pre-epithelial component, mucus-bicarbonate-phospholipid; then an epithelial component, the continuous layer of epithelial cells connected by tight gap junctions, which generates bicarbonate, mucus, phospholipids, trefoil peptides, prostaglandins, and heat shock proteins; In addition, there are other mechanisms such as cell renewal; continuous blood flow; production of nitric oxide and hydrogen sulfide; and sensory innervation [7].
Natural products are evolutionarily designed and chemically differentiated from the majority of synthesized molecules. Besides, they are capable of regulating biological systems because they are able to interact with various macromolecules [8]. Thus, natural products have played an important role in the process of development and discovery of new drugs [9], due to their wide structural diversity and because they present mostly specific and selective biological activities [10].
Among the natural products the alkaloids, biologically active secondary metabolites, that can be found in plants, animals or microorganisms, stand out. Biosynthetically, the alkaloids are derived from amino acid biosynthesis or transamination processes, and they are classified according to the amino acid that yields the nitrogen atom as well as the part of its skeleton for the synthesis of the alkaloid in question [11]. Thus, the alkaloids are compounds consisting of a basic nitrogen atom that may or may not be part of a heterocyclic ring [12].
In this article, we review studies published between January 2008 and September 2014 on alkaloids with peptic ulcer activity. The survey was conducted in databases such as SciFinder Scholar ® , Science Direct ® and PubMed ® using as keywords: gastroprotective or antiulcer or antiulcerogenic activity and alkaloids. The alkaloids cited in this review were selected according to pharmacological action demonstrated in specific experimental models to evaluate the anti-ulcer activity and/or through studies in order to discover their mechanism of action.

Rutaecarpine
The alkaloid rutaecarpine (8,13- Intravenous administration of rutaecarpine in rats at doses of 100 or 300 mg/kg significantly reduced the ulcer index and pH value when compared to the control group in ulcer models induced by both acetylsalicylic acid (ASA) and stress. ASA promotes back-diffusion of H + the mucosa and signicantly increased the ulcer index, indicting the development of hemorrhagic and non-hemorrhagic lesions of the gastric mucosa [46].
Under stress sympathetic nervous system (SNS) and parasympathetic (SNP) stimulation occurs. Sympathetic stimulation promotes arteriolar vasoconstriction, causing reduction in blood flow in mucosa, promoting local hypoxia and ischemia, generating ROS, lipid peroxidation and depletion of glutathione levels. The stimulation of SNP increases with consequent worsening motility of the gastrointestinal muscle contraction, mucosal ischemia leading to increased secretion of acid [47,48].
Calcitonin gene-related peptide (CGRP) is a neuropeptide predominantly found in capsaicin-sensitive sensory neurons, which are in abundance in the gastrointestinal tract. These nerves are involved in gastroprotection, CGRP being a potential mediator of this process, the synthesis of this neurotransmitter is regulated by transient receptor potential vanilloid subfamily member 1 (TRPV1). The beneficial effects of CGRP include increased mucosal blood flow, inhibition of gastric acid secretion, prevention of apoptosis and oxidative injury [49]. Wang et al. [46] demonstrated that the gastroprotective effect by rutaecarpine (300 or 600 µg/kg, p.o.) in models of ASA or stress-induced gastric ulcer was decreased by the pretreatment with capsaicin (50 µg/kg, p.o.) an antagonist of the TRPV1. According to studies by Aizawa et al. 2001 [50], N G -nitro-L-arginine methyl ester (L-NAME), inhibitor of nitric oxide synthase (NOS), reduces the levels of CGRP. Endogenously, NOS is inhibited by L-arginine analogue asymmetric dimethylarginine (ADMA) formed by hydrolysis of proteins in which the arginine residues are methylated from arginine methyltransferase, while being degraded by dimethylarginine dimethylaminohydrolase (DDAH) [51]. Thus, in the ethanol-induced ulcer model in rats, ruteacarpine increases the activity of DDAH reducing the levels of ADMA, enhancing NO synthesis and reducing the gastric damage [51]. Rutaercapine also present gastroprotective effects by increasing the release of CGRP by two mechanisms: TRPV1 activation and increased activity of DDHA. Pharmacological studies using the HCl/etanol-induced ulcer model displayed the anti-ulcer activity of 2-phenylquinoline at the dose of 50 mg/kg. In this model, ethanol promotes destabilization of mucus-bicarbonate-phospholipid layer, leading to retrodiffusion of H + ions, with consequent damage to epithelial cells and mast cell activation inducing the release of inflammatory mediators, that allow the migration of neutrophils to the injured area, releasing reactive oxygen species that elevate the cellular damage and result in tissular necrotic damages. The 2-phenylquinoline inhibited ulcerative lesions in 60.2% ± 7.9%, being this result significant when compared with the negative control [52,55].

Phenylquinoline
Additionally, when 2-phenylquinoline is evaluated in the pylorus ligation model, it is possible to observe changes in the gastric juice parameters, like a reduction in the volume of gastric juice and the total acidity, while gastric pH compared was increased significantly compared with the control group [52].
In addition, the involvement of nitric oxide and gastric mucus from the models used to determine contents of mucus adhered to gastric mucosa and ulcer model induced by ethanol with pre-treatment L-NAME it was observed [52].
Mucus is the first line of defense of the mucous membrane, being secreted by epithelial cells and consisting of 95% water and 5% mucin, a polymerized glycoprotein that forms a gel. This secretion is stimulated by gastrointestinal hormones such as gastrin and secretin, prostaglandin (PGE2) and cholinergic agents [6].
Nitric oxide, on the other hand, is formed from the conversion of L-arginine to citrulline by the action of nitric oxide synthase (NOS). The NO is implicated in the increase of mucus secretion, bicarbonate and blood flow in the gastric mucosa, as well as the inhibition of gastric acid secretion, providing gastroprotection and eliminating free radicals with consequent reduction of gastric lipid peroxidation [56][57][58]. After administration of L-NAME an increase of 42% in mucus production was observed and, there was a reduction in the inhibition of 53.2% to 48.4%, suggesting that at least in part, nitric oxide participates in the gastroprotection [52]. Figure 3) is an alkaloid commonly found in plants of Solanaceae family, obtained especially from the dried tobacco leaves of Nicotiana tabacum L., and the presence of this alkaloid in human matrices happens first by exposure to tobacco smoke [42,59]. Nicotine causes psychological and physical dependence when used chronically. This substance interacts with cholinergic receptors in adrenal medulla, autonomic ganglia, neuromuscular junction and brain of mammals [60] In the ASA-induced ulcer model, nicotine reduced the ulcerative lesions, probably due to an increase of the mucus secretion as reflected by the increase in pH and intragastric volume. However the nicotine did not increase the mucosal blood flow, but rather decreased it by about 18% [60].

Nicotine
Meanwhile, in another study animals were pretreated with nicotine (0.05 mg/mL in tap water) for 28 days and after treated with ethanol. There was an increase in gastric mucosal injury in the animals that received the pretreatment with nicotine, while the nicotine itself had no effect on gastric lesions. Besides, in ethanol-induced gastric mucosal injury an increase of L-arginine analogue asymmetric dimethylarginine (ADMA) levels was observed in gastric juice and the animals that received the chronic pretreatment with nicotine had an increased level of ADMA [61].
In vitro studies were also conducted, where mucosal epithelial cells were treated with nicotine (3 or 10 µmol/L) in the presence or absence of ethanol, and then the average concentration of ADMA in culture and the proportion of cells undergoing apoptosis were measured. Much higher levels of ADMA as well as increased apoptosis were observed in cells treated with nicotine and ethanol. Therefore, nicotine increases the levels of ADMA and reduced NO levels, leading to the damage caused by ethanol to the gastric mucosa [61].
To evaluate the anti-ulcer activity rohitukine several protocols were performed in an ulcer model induced by stress, where animals were treated with the alkaloid in doses of 10, 20 and 40 mg/kg orally; in the ulcer model induced by pylorus ligature, where rohitukine was tested at a dose of 20 mg/kg, with the objective of evaluating the gastric juice, peptic activity and levels of mucin; and the model of ulcer induced by ethanol. Moreover, gastrin levels, intracellular calcium ions and the activity of the H + /K + ATPase were measured, being these two last tested in vitro [67].
Rohitukine decreased the index of ulcerative lesion compared to the control group in the stress-induced ulcer model as in the ethanol model. This alkaloid reduced levels of gastric juice and free/total pepsin, blocked the activity of H + /K + ATPase, normalized gastrin levels and reduced levels of Ca 2+ in parietal cells, furthermore, it enhanced gastric mucosa defense mechanisms by increasing the levels of prostaglandin E2 (PGE2) and mucin [67].
The parietal cells are responsible for the production of gastric secretion [47]. This secretion creates a favorable medium for the conversion of pepsinogen to pepsin (an important enzyme in the protein digestion process), facilitating the absorption of iron, calcium and vitamin B12, as well as preventing bacterial overgrowth and possible infections [68].
Moreover, the parietal cells contain a small number of intracellular canaliculi and a series of tubular vesicles that have the pump H + /K + ATPase. The stimulation of these cells leads to fusion of the tubule vesicles with the membrane of the apical cell, and the activation of H + /K + ATPase, that starts to pump H + into the gastric lumen and K + into the intracellular medium. The K + in turn returns into the gastric lumen by co-transport with Cl − ions in the apical portion of the cell [47,69].
The primary stimulation of acid secretion in the gastric mucosa is performed by histamine, gastrin and acetylcholine [47]. Histamine is released from cells similar to enterochromaffin (ECL) and stimulates the parietal cell directly via H2 receptors coupled to protein Gs. Gastrin is released from the G cells and stimulates parietal cells either directly or indirectly by interacting with receptors of cholecystokinin-2 (CCK-2) present in ECL cells, inducing the release of histamine [47,69]. The acetylcholine released from postganglionic neurons, stimulate the parietal cell receptors directly or indirectly via M3, M2 and M4 receptors coupled to the inhibition of secretion of somatostatin [47, 69,70].
Somatostatin released by D cells interacts with sstR2 receptors, whereas PGE2 acts on the EP3 receptor, both located on parietal cells and coupled to Gi protein, and then they inhibit the production of adenosine monophosphate (cAMP), leading to reduction of acid secretion [47,69].

Methoxycanthin-6-one
The alkaloid 2-methoxycanthin-6-one (9-methoxy-6H-indolo[3,2,1-de] [1,5]naphthyridin-6-one, Figure 5) was isolated from the crude methanol extract of the stem wood of Quassia amara L. [71]. This alkaloid demonstrated potent actives related to male antifertility in male rats [72] and antiulcer activity [73]. To evaluate the gastroprotective activity of 2-methoxycanthin-6-one, the ulcer model induced by indomethacin was realized in doses of 12.5, 25 and 50 mg/kg, p.o. and 1, 2 and 4 mg/kg, i.p. [73]. Indomethacin is a nonselective cyclooxygenase inhibitor that impairs the synthesis of prostaglandins. As a result of the inhibition of PGE2 and PGI2 synthesis there is a decrease in gastric mucus and bicarbonate production and changes in blood flow, thus, reducing the protective barrier of the normal mucosa and also the ability to repair mucosal cells [74]. The results shown that 2-methoxycanthin-6-one produced a dose-dependent gastroprotective effect on indomethacin-induced gastric lesions in rats [73].
Moreover, the pylorus ligature model was used wherein was performed using the dose which produced better effect 4 mg/kg i.p in ulcer model induced by indomethacin. In this model, cimetidine, a histaminergic antagonist, was used, in order to determine whether the mechanism of action of 2-methoxycanthin-6-one has any participation of the histaminergic pathway. 2 Methoxycanthin-6-one decreased gastric secretion, however, in the presence of cimetidine, this effect was reduced, demonstrating that is likely an alkaloid H2 antagonist [73].

Chelerythrine
Chelerythrine chloride (1,2-dimethoxy-N-methyl(1,3)benzodioxolo(5,6-c)phenanthridinium chloride, (Figure 6) is an alkaloid commonly found in the Papaveraceae and Rutaceae families which can be classified as a quaternary benzo[c]phenanthridine alkaloid [75]. Among its reported biological activities, there are anti-inflammatory effects [76] and interaction with certain proteins such as protein kinase C (PKC) and mitogen-activated protein kinase phosphatase-1 [77]. Li et al. [75] evaluated the gastroprotective effect of chelerythrine against ethanol-induced gastric mucosal lesions. A pre-treatment with chelerythrine at doses of 1, 5 and 10 mg/kg, p.o. was realized in mice for four days, followed by administration of absolute ethanol (0.2 mL/animal). A decrease of the ulcerative level in a dose dependent manner was observed when compared to the control group [75]. Gastric juice acidity and mucus production were also evaluated. Animal groups pre-treated with this alkaloid showed a significant increase of pH when compared with the control group.
From the histopathological studies it was observed that pre-treatment with chelerythrine resulted in a reduction in ulcer area, and reduction or absence of submucosal edema and leucocytes infiltration. Furthermore, a reduction in the overproduction of mucus and NO in the serum and gastric tissues was observed [75]. This overproduction of NO occurs during innate immune system responses when activated macrophages produce NO to play its bactericidal activity [78].
Besides, by immunohistochemistry analysis and western blot it was possible to observe an effect of chelerythrine on protein expression of NF-κB p65 in gastric tissue and the effect of chelerythrine on cytokines was determined by ELISA [75].
Cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) and IL-10 play an important role in acute inflammation and in the maintenance and regulation of gastric mucosal damage [79,80]. TNF-α has the capacity to induce fever, stimulates the production of acute-phase proteins by the liver and causes endothelial cell activation [55], while IL-6 causes high levels of active lymphocytes, neutrophils and monocytes/macrophages in the inflamed area [75].
The expression of these pro-inflammatory mediators may be increased by the activation of NF-κB subunits (p65 and p50) and can be reduced by the inactivation of NF-κB whereby PKC phosphorylates the subunit 65 [81,82]. Chelerythrine reduced the levels of TNF-α and IL-6, pro-inflammatory cytokines, in serum and tissue of rats exposed to ethanol as well as reduced levels of p65 subunit of NF-κB, showing the initial stages of inflammation action [75].
To evaluate the gastroprotective activity of this alkaloid, initially the gastric ulcer model induced by ethanol was performed in which pirplatine was tested at doses of 4.5 mg/kg, p.o. Additionally, the gastric acid secretion from the pylorus ligation was evaluated, where pirplatine was tested at doses of 4.5 and 15 mg/kg i.d. This evaluation was undertaken to determine the gastric mucous, the levels of reduced glutathione and evaluate the activity of the H + /K + ATPase pump [95].
Piplartine reduced the rate of ulcerative lesions compared to the control group, and did not affect the levels of mucus, however, it increased the levels of GSH, reduced the activity of H + /K + ATPase pump, reduced the volume and total acidity of the gastric juice and reversed the stimulatory effect of pentagastrin (CCK2 receptor agonist) on gastric acid secretion, demonstrating that piplartine is a CCK2 receptor antagonist [95].
The gastric mucosa is constantly assaulted by various endogenous and exogenous factors [96], however it has several defense mechanisms, such as the mucus layer that acts as a physical barrier [6] and reduces the damage caused by oxidative stress [97], reduced glutathione, that acts directly in the oxidizing system [98]. Piplastine has gastroprotective effects by increasing levels of GSH and its anti-secretory effect [95].

N-Isopropylmethylanthranilate
N-isopropylmethylanthranilate (NIM, Figure 8), was initially identified in the essential oil of Choisya ternata Kunth (Rutaceae) by the gas chromatography and gas chromatography-mass spectrometry techniques, albeit in small amounts, then e NIM was synthesized in order to obtain sufficient amounts to test its possible biological activities [99]. NIM is a volatile alkaloid and due to this property, it were tested in animal models as a potential anxiolytic, antidepressant and antinociceptive drug [99,100]. After these studies, Radulović and colleagues [101] hypothesized that due to the antinociceptive activity observed in this alkaloid it could have anti-inflammatory activity and consequently have anti-ulcer effects since peptic ulcer is an inflammatory phenomenon.
Initially, Radulovićet et al. [101] evaluated the effects of NIM (200 mg/kg, p.o.) on the integrity of the gastric mucosa to verify a possible ulcerogenic effect of these substances. However the alkaloid did not alter the architecture of the gastric tissue. After that, the gastroprotective activity of NIM (50, 100 and 200 mg/kg, p.o.) in animal models of ulcers induced by NSAIDs (diclofenac 80 mg/kg, p.o.) and ethanol was investigated. All evaluated doses (p < 0.05) of the two substances protected the stomach from diclofenac and ethanol-induced lesions [101].

N-Methylmethylanthranilate
The alkaloid N-methylmethylanthranilate (NMM, Figure 9) is considered an important marker of the essential oil of Citrus recutita, found in species of the Rutaceae family, particularly in the Rutoideae and Aurantioideae subfamiles [99]. In the Choisya ternata Kunth (Rutaceae) species this alkaloid was found in small quantities by chromatographic techniques, however, it is readily synthesized, and it can thus be obtained in sufficient quantities to test its pharmacological effects [99]. The NMM has demonstrated antinoceptive action in a dose-dependent manner [99], as well as a significant anxiolytic and antidepressive effect [100]. Moreover, it showed gastrorprotective effects in models of ulcer induced by ethanol and diclofenac in doses of 50, 100 and 200 mg/kg, po, when compared to the control group [101].
A study was conducted by Almeida and colleagues [109] to investigate the mechanism involved in the gastroprotective activity of canthin-6-one (20 mg/kg, p.o.). The results suggest that the alkaloid has gastroprotective effects (against ethanol and indomethacin-induced ulcer) by stimulating nitric oxide production, acting by modulating the levels of pro-inflammatory cytokines (IL-8) and decreasing the activity of myeloperoxidase (MPO) and lipid peroxidation, suggesting a possible antioxidant activity.
PEG is a substance with antioxidant activity (in vitro DPPH assay) and antibacterial activity (agar diffusion assay). It also has cytotoxic activity toward different strains of cancer, with better results seen in tests performed with Sp2/O-Ag14 cells (IC50 = 50 mg/mL to >100 mg/mL) [123,126].
Peganine (20 mg/kg, p.o.) also protected the stomach from the ulcerative lesions induced by ethanol (89.41% of protection), non-steroidal anti-inflammatory drugs (NSAIDs) (58.50%) and pylorus ligature (62.50%). The mechanism of action of the gastroprotective activity of PEG involves the reduction of gastric acid secretion that was verified by biochemical parameters of gastric juice (in vivo) and inhibitory activity of the proton pump (in vitro). Furthermore PEG acted by modulating the levels of PGE2 and displayed antioxidant activity an in vitro DPPH assay [125].

Conclusions
In summary, this review demonstrates the great importance of stimulating studies with natural products, especially alkaloids, for the discovery of new therapeutic alternatives in healing of peptic ulcer due to these metabolites' broad and differentiated mechanisms of action, thus potentially benefiting the population in general by promoting access to medicines and offering a better quality of life.